medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256299; this version posted April 30, 2021. The copyright holder for this preprint 1 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Metabologenomic approach reveals intestinal environmental features associated with barley- induced glucose tolerance improvements in Japanese cohort: a randomized controlled trial Yuka Goto1¶, Yuichiro Nishimoto2¶, Shinnosuke Murakami2,3, Tatsuhiro Nomaguchi2, Yuka Mori2, Masaki Ito2, Ryohei Nakaguro2, Toru Kudo2, Tsubasa Matsuoka1, Takuji Yamada2,4, Toshiki Kobayashi1*, Shinji Fukuda2,3,6 * 1 Hakubaku Inc., Chuo, Yamanashi, Japan. 2 Metabologenomics Inc., Tsuruoka, Yamagata, Japan. 3 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan. 4 Department of Life Science and Technology, Tokyo Institute of Technology, Meguro, Tokyo, Japan. 5 Intestinal Microbiota Project, Kanagawa Institute of Industrial Science and Technology, Kawasaki-ku, Kawasaki, Kanagawa, Japan. 6 Transborder Medical Research Center, University of Tsukuba, Tsukuba, Ibaraki, Japan. ¶ These authors contributed equally to this work. *Corresponding author: [email protected], [email protected] Running title: Intestinal environments associated with barley induced blood parameter improvements NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256299; this version posted April 30, 2021. The copyright holder for this preprint 2 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Abstract Consumption of barley has been known to exert beneficial effects on metabolic disorders; however, it has also been reported that there are inter-individual differences in these responses. Recent evidence has suggested that these individual differences are mediated by the gut microbiota. Therefore, in the present study, we aimed to understand the relationship between the intestinal environment, including gut microbiota, and metabolic disorders. A randomized controlled trial in Japanese subjects with 4-week consumption of barley or control food was conducted. In this study, we analyzed the intestinal environment, including microbiota and their metabolites, and blood parameters were assessed collectively. We found that microbial genera Blautia and Agathobacter belonging to Lachnospiraceae, and fecal metabolites such as azelate were increased 1.31-fold, 1.84-fold, and 1.48-fold after barley consumption, respectively. Furthermore, the subjects whose glucose tolerance were slightly impaired showed improvement in their glucose tolerance index following the barley consumption. Additionally, the analysis showed that the increase in the abundance of the Anaerostipes was correlated with the improvement in the glucose tolerance index. Our findings indicate that the effects of barley consumption for glucose tolerance are partly defined by the intestinal environment of consumers, providing a quantitative measurement of the dietary effect based on the intestinal environment. medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256299; this version posted April 30, 2021. The copyright holder for this preprint 3 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Introduction Barley is one of the first domesticated grains and has been cultivated for about 10,000 years. Recent studies have revealed that barley has the potential to improve certain blood parameters such as glucose tolerance and total blood cholesterol (1, 2). As barley contains a high amount of β-glucan, a type of soluble fiber, its effect on these blood parameters has been attributed to its high viscosity. It has been reported that barley-derived β-glucan conjugates with sugars and lipids in the human gastrointestinal lumen to inhibit their digestion and absorption (3, 4). In addition, a previous study showed that barley consumption increases Prevotella/Bacteroides ratio in barley-consuming healthy Swedish subjects with improved glucose tolerance (5). These reports indicate that β-glucan affects blood glucose levels in two ways: directly by binding to sugars and indirectly through changes in the intestinal environment. Despite these studies, the complete picture on the underlying relationship between barley intake and glucose intolerance is yet to be revealed, as many reports suggest that there are multiple confounding factors. First, genetic background affects the blood parameters. It has been reported that the primary symptom in the early stage of diabetes is different in populations from different backgrounds. For example, Caucasians show insulin resistance, whereas East Asians show a decrease in insulin secretion (6). Second, it has been suggested that the effect of barley is partly due to the functions of the intestinal microbiome (5); however, within the same species of bacteria, the existence of different strains unique to each country has been implied. It has also been proposed that the differences in these strains can possibly lead to differences in the phenotypes. For instance, various strains of Prevotella copri have been observed in multiple countries, and their functions are also dissimilar (7). Moreover, there have been no studies reporting the effect of barley on the intestinal environment and glucose tolerance index in Asian populations, such as the Japanese, who are known to have unique microbiomes (8). As explained above, the effect of barley intake on the intestinal microbiome and metabolome profiles is still unclear. To elucidate the mechanisms through which barley improves the blood parameters through the intestinal environment in Japanese subjects, a randomized double-blind controlled trial was conducted. The intestinal environment was evaluated using a metabologenomics approach, which combines mass spectrometry-based metabolome and high-throughput sequencing-based microbiome analyses. medRxiv preprint doi: https://doi.org/10.1101/2021.04.29.21256299; this version posted April 30, 2021. The copyright holder for this preprint 4 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Results The effect of barley intake on blood parameters We conducted a randomized, double-blind controlled trial in 19 Japanese subjects (Fig 1). Baseline clinical characteristics were similar in primary and secondary outcomes including blood glucose area under the curve (AUC) and incremental AUC (iAUC), insulin AUC and iAUC, fasting blood glucose and insulin and stool frequency in both groups (Table S1). First, we statistically tested each primary and secondary outcome; however, there were no significant differences observed in any outcomes (Table 1). To investigate the beneficial effect of barley, parameters obtained from the clinical blood test, which was performed at six different time points, were evaluated through multiple testing (Friedman test followed by Nemenyi’s post-hoc test). There were significant differences in sodium, total cholesterol, and total protein levels (Fig 2, q < 0.10, False Discovery Rate (FDR) corrected). Next, the post-hoc test revealed a significant decrease at T3 (end of test food intervention period) in total protein and cholesterol levels compared to C1 and T1, respectively. For sodium, a significant decrease was observed at C3 and T2 compared to T1. Effect of barley intake on intestinal microbiome and metabolome profiles To evaluate the effect of barley on intestinal microbiome and metabolome profiles, we performed 16S rRNA gene-based microbiome analysis and CE-TOFMS-based metabolome analysis. Multidimensional scaling with beta diversity showed that the plots from each subject were clustered in both microbiome and metabolome profiles (Fig 3). Comparison of inter-individual distances and intra-individual distances showed significant differences (Wilcoxon rank sum test, p = 9.90*10-180 and p = 3.99 * 10-116 at microbiome and metabolome profile, respectively), suggesting that the individual difference was larger than the influence of barley or control food consumption (Fig 3C, F, Fig S1). We next performed the Wilcoxon signed-rank test for the relative abundance of each microbe and the relative area of each metabolite, to further investigate the effect of barley consumption on the intestinal environment. The comparison was performed between pairs of two different sets; T1–T3 and C3–T3 (Fig 4). Some genera showed significant differences in their relative abundances (p < 0.05, not corrected), such as Blautia and those belonging to the family Lachnospiraceae in T1–T3 comparison; however, these differences were only observed as significant without multiple testing correction (Table S2). Additionally, several metabolites showed significant increases (e.g. azelate and imidazole propionate) and decreases (e.g. paraxanthine and 6-hydroxynicotic acid) in their relative area; however, the significance was only observed without multiple testing